Neurons communicate with various target cells by the release of neurotransmitters. Neurotransmitters exert their effects by binding to sites called receptors that are located on the extracellular surface of their respective target cells. Numerous neurotransmitters have been identified, one of which is dopamine. Schizophrenia and Parkinson's disease are believed to be related at least in part to disturbances in the operation of dopamine and its receptors.
A major function of a neurotransmitter receptor is to transmit information into the interior of a target cell, causing various rapid responses of the cell. One of the major classes of receptors is known as G protein-coupled receptors. They act by coupling to a family of signal-transducing proteins located on the cytoplasmic surface of the plasma membrane. These proteins are called G proteins because they bind guanine nucleotides when activated by neurotransmitter receptors. After activation, G proteins are able to regulate a variety of cellular events, including the activity of ion channels and enzymes.
Several of the genes that code for mammalian G protein-coupled receptors have been cloned. These include the alpha-2 and beta-2 adrenergic receptors, five subtypes of muscarinic acetylcholine receptors, three subtypes of serotonin receptors, the substance K receptor, and a rat dopamine D2 receptor. All of the G protein-coupled neurotransmitter receptors cloned to date have a predicted structure having seven transmembrane domains and several regions of strong homology, suggesting that they may have arisen from a common ancestral gene.
The cloning of subtypes of some of these neurotransmitter receptors indicates a molecular basis for pharmacological heterogeneity. For example, several subtypes of muscarinic, serotonergic, and adrenergic receptors have now been cloned. See Bonner et al., Science, 237: 527-532 (1987); Bonner et al., Neuron, 1: 403-410 (1988); Peralta et al., EMBO J., 6: 3923-3929 (1987); Kubo et al., Nature, 323: 411-416 (1986); Julius et al., Science, 241: 558-564 (1988); Fargin et al., Nature, 335: 358-360 (1988); Kobilka et al., Science, 228: 650-656 (1987); and Dixon et al., Nature, 321: 75-79 (1986), all of which are incorporated herein by reference. These receptor subtypes are expressed in distinct regions of the brain and body.
The cloning of the muscarinic acetylcholine receptors provides an example of how the general technique of homology screening was applied to the identification of a family of genes encoding a G protein-coupled receptor. See Bonner et al., Science, 237: 527-532 (1987) and Bonner et al., Neuron, 1: 403-410 (1988), both of which are incorporated herein by reference.
In the work reported in Science, the authors made a probe from the highly conserved transmembrane region near the 5' region of the porcine brain muscarinic receptor gene with certain modifications. The probe was used to screen a rat cerebral cortex cDNA library of clones in the pCD mammalian expression vector. The clones were characterized before isolation by using Southern blots of portions of the library. Dilutions were done to obtain a single band by Southern blotting. The cDNA inserts were transfected into mouse fibroblast (A9) or COS-7 cells. The transfected cells were assayed for the ability of the cell membranes to bind a muscarinic antagonist. The authors found four different muscarinic receptor genes.
The Neuron article reports the cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes in an approach similar to that in the Science article. A human genomic library was screened using the rat ml receptor gene as a probe, which identified the human m5 receptor gene. The homologous rat gene was isolated from a rat genomic library, using a portion of the human gene as a probe. The coding portion of the human gene was inserted into the plasmid pCD-PS, a mammalian expression vector that utilizes an SV40 promoter, and expressed in CHO cells.
On the basis of functional and pharmacological data, central dopamine receptors have been divided into D1 and D2 subtypes. Dopamine D1 and D2 receptors stimulate and inhibit adenylate cyclase, respectively, by coupling to different G proteins. See Kebabian et al., Nature, 277: 93-96 (1979), incorporated herein by reference. D2 receptors are known to be abundant within various forebrain regions innervated by dopaminergic neurons projecting from the mesencephalon. See Bjorklund and Lindvail, "Dopamine Containing Systems in the CNS, " in Handbook of Chemical Neuroanatomy Vol. 2: Classical Transmitters in the CNS, Bjorklund and Hokfelt, eds., (Elsevier, Amsterdam, 1984), pp. 55-122; Fuxe et al, "Dopaminergic Systems in the Brain and Pituitary," in Basic and Clinical Aspects of Neuroscience, Fluckiger et al., eds., (Springer Verlag & Sandox, Heidelberg, 1985), pp. 11-25; Anden et al., Life Sci., 3: 523-530 (1964); Anden et al., Life Sci., 4: 1275-1279 (1965); and Dahlstrom et al., Acta. Physiol. Scand., 62: 485-486 (1964), all of which are incorporated herein by reference. D2 receptors have also been shown to be presynaptically located on dopaminergic neurons, where they modulate dopamine release. See Roth, Ann. N.Y. Acad. Science, 430: 27-53 (1984) and White et al., J. Pharm. Exp. Ther., 231: 275-280 (1986), both of which are incorporated herein by reference.
Abnormalities in central dopaminergic function have been implicated in the pathophysiology of schizophrenia and Parkinson's disease. See Stevens, Arch. Gen. Psychiatry, 29: 177-189 (1973); Synder, Amer, J. Psychiatry, 133: 197-202 (1976); and Hornykiewicz, Wien. Klin. Wochenschr., 75: 309-312, all of which are incorporated herein by reference. Schizophrenia is treated with antipsychotic drugs which block central D2 receptors, and Parkinson's disease is treated by drugs which lead to their stimulation. See Baldessarini, "Drugs and the Treatment of Psychiatric Disorders," in The Pharmacological Basis of Therapeutics, Gilman et al., eds., (Macmillan, N.Y., 1985) pp. 387-445; Bianchine et al., "Drugs for Parkinson's Disease, Spasticity, and Acute Muscle Spasms," in The Pharmacological Basis of Therapeutics, Gilman et al., eds., (Macmillan, N.Y., 1985) pp. 473-490; Iversen et al., Science, 188: 1084-1089 (1975); and Seeman et al., Nature, 261: 717-719 (1976), all of which are incorporated herein by reference.
Prolonged treatment with antipsychotic agents is associated with extrapyramidal (motor) side effects such as tardive dyskinesia. Since the motor side effects are believed to be mediated by receptors located within the striatum, and since antipsychotic activity is believed to be mediated by sites within limbic and cortical regions, investigation of a potential heterogeneity of D2 receptors between these brain regions has become a focus for drug development. See Borison et al., Brain Res. Bull., 11: 215-218 (1983); Leonard et al., Biochem. J., 248: 595-602 (1987); and Baldessarini et al., Annu. Rev. Neurosci., 3: 23-41 (1980), all of which are incorporated herein by reference.
Dopamine is the major catecholamine present in retina, where its synthesis and release is stimulated by light. Invone et al., Science, 202:901-902 (1978), incorporated herein by reference. As in the brain, retinal dopamine receptors have been divided into two subtypes, based on their pharmacological and functional properties. See Elena et al., Curr. Eye Res., 8:75-83 (1989) and Qu et al., J. Pharm. Exp. Ther., 248:621-625 (1989), both of which are incorporated herein by reference. Dopamine D1 receptors stimulate adenylate cyclase by coupling with the G-protein Gs, and D2 receptors inhibit adenylate cyclase by coupling with the G-protein Gi. Various drugs discriminate between these receptors; for example, SCH23396 has higher affinity for D1 receptors, while substituted benzamides, such as sulpiride and raclopride, have higher affinity for D2 receptors. See Qu et al., op.cit. and Hall and Wadel, Acta Pharmacol et toxicol., 58:368-373 (1986), incorporated herein by reference.
A dopamine D2 receptor from rat brain was recently cloned, sequenced, and expressed. See Bunzow et al., Nature, 336: 783-787 (1988), incorporated herein by reference. The article reports the use of the hamster beta-2 adrenergic receptor gene as a hybridization probe to screen a rat genomic library under low stringency hybridization conditions. The authors found a clone having a fragment with a high degree of nucleotide identity to one of the transmembrane domains of the gene. This fragment was used to probe a rat brain cDNA library under high stringency hybridization conditions. A fragment of about 2.5 kB was isolated. Using this cDNA as a probe in a Northern Blot analysis of mRNA from rat brain, the authors showed that the cDNA was nearly full-length. The cDNA was determined to be 2,455 bases in length. Comparison with the genomic clone showed the presence of at least one intron in the coding region. The tissue distribution of the encoded mRNA was examined by Northern Blot analysis and found to be very similar to that for the dopamine D2 receptor. The cDNA was cloned into a eucaryotic expression vector and transfected into a mouse fibroblast cell line. Stable transfectants were isolated. The cell membranes were shown to bind a D2 ligand. The paper provided a deduced amino acid sequence for the receptor. The authors concluded that the structural features of the protein showed it to belong to the family of G protein-coupled receptors.
Prior to the present invention, the scientific literature does not report the isolation, cloning, and expression of a gene encoding a human dopamine D2 receptor. Such DNA and cell lines expressing it have several important scientific and pharmaceutical applications. First, the cloning and expression of human dopamine D2 receptor genes would permit the receptors to be studied in isolation from other related dopamine receptors.
Second, although there is substantial species homology for all of the G protein-coupled receptors that have been cloned to date, all of the receptors have differences in their predicted protein sequences. These differences are likely to translate into differences in their pharmacology. Thus, for example, it would be highly desirable to use human dopamine D2 receptors rather than the rat dopamine D2 receptors for drug development purposes.
Third, the fact that pharmacological subtypes of some of the other G protein-coupled receptors, such as the muscarinic receptor, have been found to be encoded by different genes, together with certain pharmacological data reported herein, suggests that dopamine receptors may consist of multiple genetic subtypes. Cloning and expression of the genes for those subtypes would permit the study of subtle differences in their pharmacology.